The ability to effectively isolate rare cells and identify subset of cell characteristics could provide significant prognostic and therapeutic implications ranging from cancer diagnostics to stem cell therapies. The rare nature of these cells imposes particular challenge on the functionality and robustness of the separation and analysis methodology. Nanotechnology could provide novel and practical solutions to meet such challenge. The broad objective of this proposal is to develop superior nanoparticle reagents in combination with a unique separation scheme that could be applied for highly effective and multiplexed separation of rare cells. These nanoparticle agents will be based on the highly fluorescent magnetic nanoclusters (HFMN) with superior fluorescent properties and precisely tunable magnetic moments. HFMN will provide an excellent platform technology that not only allows optimized magnetic control for effective separation of rare cells, but also facilitates multiplexed cell separation using a specifically designed external magnetic field gradient control. In addition, the superior fluorescent signal of HFMN will allow simultaneous tracking and in situ biomedical analysis for cell type identification and for diagnostic or therapeutic evaluations. The specific aims are: 1) Synthesize HFMN with optimal fluorescent brightness and tunable magnetic properties. 2) Apply HFMN for efficient rare cell separation and identification in whole blood samples. 3) Demonstrate the advanced capability of HFMN in combination with a specifically designed external magnetic field gradient control for multiplexed separation and identification of rare cells in whole blood samples. Such high quality multifunctional nanoparticles and the unique magnetic separation scheme when made commercially available, could significantly advance the current technology in rare cell separation and analysis, and ultimately contribute to improved cellular diagnosis and therapy for many patients. PUBLIC HEALTH RELEVANCE: The goal of this research is to develop novel nanoparticle reagents, the highly fluorescent magnetic nanoclusters, and a specifically designed magnetic separation scheme for highly effective and multiplexed separation and identification of rare cells. Success in achieving this goal could significantly advance the current technology in rare cell separation and analysis including circulating tumor cells and therapeutic cells such as T-cells and stem cells, and ultimately contribute to improved cellular diagnosis and therapy for many patients.